Colon cancer is the third most common malignancy around the world. Surgery, chemotherapy, and radiotherapy are generally used to treat colon cancer, but no effective therapy for advanced colon carcinoma is available. Therefore, there is a need to identify other therapeutic agents against this disease. Magnolol, a hydroxylated biphenyl compound present in Magnolia officinalis, exerts anticancer potential and low toxicity. Emerging evidence has suggested that activation of AMP-activated protein kinase (AMPK), a potential cancer therapeutic target is involved in apoptosis in colon cancer cells. However, the effects of magnolol on human colon cancer through activation of AMPK remain unexplored. In this study, we explored whether magnolol exerts an antiproliferative effect, and induces apoptosis in HCT-116 human colon cancer cells. Magnolol displayed several apoptotic features, including propidium iodide labeling, DNA fragmentation, and caspase-3 and poly(ADP-ribose) polymerase cleavages. We showed that magnolol induced the phosphorylation of AMPK in dose- and time-dependent manners. The selective AMPK inhibitor compound C abrogated the effect of magnolol on AMPK activation, suppression of proliferation, and caspase-3 cleavage. Magnolol downregulated expression of the antiapoptotic protein Bcl2, upregulated expression of pro-apoptotic protein p53 and Bax, and caused the release of mitochondrial cytochrome c. Magnolol-induced p53 and Bcl2 expression was abolished in the presence of compound C. Magnolol inhibited migration and invasion of HCT-116 cells through AMPK activation. These findings demonstrate that AMPK mediates the anticancer effects of magnolol through apoptosis in HCT-116 cells.